The invention relates to peptides that bind to fluorescent dyes, termed xe2x80x9cfluorettesxe2x80x9d, and to methods of making and using the fluorettes. In particular, the fluorettes can be used in detection and assay systems in vitro and in vivo.
Fluorophore dyes, due to their exquisite sensitivity and ease of use, are widely used in numerous approaches in fluorescent microscopy, flow cytometry and other detection systems (Haugland. Handbook of fluorescent probes and research chemicals (sixth edition). Molecular Probes, Inc., Eugene Oreg. (1996) and ref. therein).
Detection of proteins in living cells using fluorescence approaches has been accomplished in a variety of settings. For instance, it is possible to use ligands (or naturally-derived antibodies) conjugated directly or indirectly to fluorophores as probes of the expression levels of nearly any given surface-expressed protein on living cells. In some particular cases for proteins within cells, it is possible to use permeable ligands for individual target proteins. In these cases the ligand is either self-fluorescent, becomes fluorescent upon binding or is conjugated to fluorescent adducts. In other cases, it has been possible for many years to genetically fuse reporter enzymes such as xcex2-galactosidase, xcex2-glucuronidase, and xcex2-glucosidase to proteins and use a fluorogenic dye, acted upon by the reporter enzyme(s), to assay enzymatic activity on a cell by cell basis (Nolan et al. Proc. Natl. Acad. Sci. U.S.A. 85:2603-2607 (1988); Lorincz et al. Cytometry 24:321-329 (1996); Krasnow et al. Science. 251:81-85 (1991). Other systems, including xcex2-lactamase (Zlokarnik et al. Science 279:84-88(1998)) build upon those findings by applying dyes with increased cell permeability or having radiometric fluorescent qualities that might have advantages in some applications. In recent years proteins with inherent fluorescence, such as Green Fluorescent Protein (Welsh and Kay. Curr. Opin. Biotechnol. 8:617-622 (1997); Misteli and Spector. Nat. Biotechnol. 15:961-964 (1997)) have become widespread in their application owing to ease of use, the availability of mutant proteins with differing spectral qualities in either excitation or emission, and the relative non-toxicity of the approach.
However, in the aforementioned cases the approaches are limited by a need to genetically fuse a relatively bulky reporter protein to the molecule under study. This can have detrimental consequences to the functionality of the protein in question or interfere mechanistically with cellular constituents with which the protein interacts. While it would be best to directly measure a given target protein using a specific fluorescent dye that recognized any given target moiety, no technology exists as yet to create such reagents.
There is a need, therefore, to develop approaches that provide the building blocks for specific biomaterial detection.
The invention provides peptides that bind to fluorophore dyes. In one aspect of the invention the peptides are made of naturally occuring amino acids, non-naturally occurring amino acids, or combinations thereof.
In another aspect of the invention, methods are provided for isolating and identifying peptides that bind to fluorophore dyes. The method comprises creating and screening peptide libraries that bind to fluorophores.
In another aspect of the invention, methods are provided for increasing the binding affinity of the fluorette for a fluorophore.
In a further aspect of the invention are provided complexes of fluorettes bound to fluorophore dyes. The binding of the fluorophore by the fluorette may alter the excitation and/or the emission spectrum of the fluorophore.
In an additional aspect, the present invention provides methods for detecting a fluorette by binding a fluorette to a fluorophore dye and detecting the fluorette/fluorophore dye complex.